Culture Media and Inoculation Methods

LAB.
CULTURE MEDIA AND INOCULATION METHODS
Topics: Culture media used in Medical Microbiology Labs, Methods of Culturing

Microorganisms, Properties of bacterial colonies and colony varieties on media.
Media: A culture media is a special medium used in microbiological laboratories to grow
different kinds of microorganisms. They are composed of different nutrients that are essential
for microbial growth. Since there are many types of microorganisms, each having unique
properties and requiring specific nutrients for growth, there are many types based on what
nutrients they contain and what function they play in the growth of microorganisms.
Classified according to three properties
A. Physical state
B. Chemical composition
C. Functional property
A. Physical state
a) Liquid media: Water-based solutions
that are generally termed “Broth”.
They are used as enrichment media and
used for biochemical tests. The growth
can be observed as turbidity
b) Semi-solid media: Contain low

percentage (<0.5%) of agar, which can
be used for motility test.
c) Solid media: They contain high percent (1-5%) of agar, which enables the formation of
discrete colonies. Colony morphology, pigmentation, hemolysis can be seen. They can be
used to prepare pure culture.
LAB. CULTURE MEDIA AND INOCULATION METHODS
B. Chemical content
a) Synthetic media: contain pure organic and inorganic compounds that are chemically
defined. They are used for special studies such as metabolic requirements.
b) Non-synthetic or complex media: Complex or enriched media contain ingredients
that are not chemically defined or pure (i.e. animal extracts).
C. Functional property of growth media

a) General Purpose / Basic media:
General Purpose Media is a
medium that provides enough
nutrients, which will be utilized
for the growth, by most of the
microorganism. It allows a wide
variety of microorganisms to
grow.
Ex:Nutrient-agar
b) Enriched media: Contain simple nutrients and additional requirements such as:
blood, serum, peptones, and vitamins. Support the growth of a wide variety of
organisms, including fastidious and pathogenic organisms.
Ex: Chocolate agar, Blood agar.
c) Selective media: This media contain substances that inhibit the growth of some
microorganisms and allow others to grow. Addition of substances such as bile
salt, dyes, and antibiotics inhibits the growth of unwanted microorganisms.
d) Differential media:
Used to differentiate microorganisms by using indicator or dyes.
e) Transport media: Transport medium are special media formulated to preserve a

specimen and minimize bacterial overgrowth from the time of collection to the
time it is received at the laboratory to be processed.
Many cultures media are both differential and selective: EMB agar, MacConkey agar,
SS agar, Mannitol Salt Agar,Hektoen Enteric Agar.
Methods of Culturing Microorganisms

Culturing is used
✓ to enrich the number of microorganism
✓ to select for certain organism while suppressing others
✓ to differentiate among different kinds of microorganisms
Remember that aseptic technique is essential!
1. Broth cultures: Broth cultures are used where individual colonies of bacteria of fungi,
growing on a solid surface.
• Remove the cotton or plug from the mouth of the liquid medium.
• Pass the mouth of the tube through the flame.
• Cool the loop close to the flame and on an empty side of the tube.
• Touch the loop to the bacterial colonies grown in culture plates, immerse into the
liquid medium.
• If the material is solid, crush the sample dragging the loop onto the tube wall.
• Carefully remove the loop from the media, do not splash the liquid material around,
carefully flame the loop, keep it sterile and leave it in the work area.
• If the sample is cultured from the solid medium to the liquid medium by the swab, the
colony is taken by swab from the solid medium. There is no need to sterilize the swab,
because the swabs are sterile. Once the colony is sucked into the dehydrated medium,
the swab is rolled back to the edge of the tube. The material on the swab is left in the
medium.
• If the specimen is taken with a swab, the swab is discarded after the process.
Solid Media Culture: All culture media must be checked visually before use for
contamination, significant physical imperfections (for example, uneven distribution of media,
variable amounts of medium in petri dishes/ tubes/ bottles, color, gross deformation of the
surface on the media) and expiry date. Culture media should have an identifiable batch or
quality control number and have passed QC tests before use. Plates that are beyond their
expiry date, contaminated plates, and broth media appearing unusually turbid should be
discarded.
a. Single Colony Streaking” technique is used to isolate a pure strain of microorganism,
often bacteria.
• a sterile loop is used to 'serially dilute' the
material around the agar plate so that well
isolated individual colonies can be detected.
The loop is sterilized between each set of
streaks so the amount of material in each set
of streaks is progressively lessened. Well
isolated colonies form in the last one or two
sets of streaks
• All culture plates and containers must be labeled to identify the patient name or laboratory
number or barcode, and date.
• Flame the loop for sterilization and cool the loop in an empty area of the solid
medium. Touch the loop to the bacteria that grows on agar plate.Streak the 1st
sector (about 1/3 of the plate) with zigzag movements.
• After the first sector is streaked, the

inoculating loop is sterilized and an
inoculum for the second sector is
obtained from the first sector.
• A similar process is followed for
streaking the third sector, and the fourth
sector.
• Thus this is essentially a dilution
process. Eventually, very few cells will
be on the loop. These develop into
separate colonies.
Colony types seen on culture plates

Bacteria grow on solid media as colonies. A colony is defined as a visible mass of
microorganisms all originating from a single mother cell, therefore a colony constitutes a
clone of bacteria all genetically alike
a. S (Smooth) Colony: Rounded colonies with smooth edges. Eg: Staphylococcus
aureus
Fig. S (smooth) colony

b. R (Rough) Colony: Non-uniform colonies with indented edges and wrinkled

appearance.
Exp: Bacillus anthracis, Mycobacterium tuberculosis
R (rough) colony
c. M (Mucoid) Colony: Sticky, smooth, bright viscous or sticky colonies
Exp: Klebsiella pneumoniae
M (mucoid) Colony
EXPECTATIONS:
In this practical time, you are expected to learn types of media, the methods of culturing in
media such as solids and liquid media, and the colony characteristics of bacteria.
USED MATERIAL:
- Loop
- Gloves
- Media

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LAB.

CULTURE MEDIA AND INOCULATION METHODS

Topics: Culture media used in Medical Microbiology Labs, Methods of Culturing

b) Semi-solid media: Contain low

C. Functional property of growth media

e) Transport media: Transport medium are special media formulated to preserve a

Methods of Culturing Microorganisms

• After the first sector is streaked, the

Colony types seen on culture plates

Fig. S (smooth) colony

b. R (Rough) Colony: Non-uniform colonies with indented edges and wrinkled

Exp: Bacillus anthracis, Mycobacterium tuberculosis

c. M (Mucoid) Colony: Sticky, smooth, bright viscous or sticky colonies

Exp: Klebsiella pneumoniae

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